This revised application seeks to continue studies on identification and repair of a number of damaged bases produced by the reaction of DNA with reactive oxygen species (ROS). A variety of base lesions have been identified in synthetic DNA oligomers exposed to ROS. The identifications were accomplished on modified, but otherwise still intact, oligomers. Several novel lesions were identified by this approach including many double lesions, i.e., lesions occurring on two adjacent or nearby bases. Double lesions appear to be a common consequence of free radical-initiated DNA damage. This amended application would extend the project into three new areas as were described in the original renewal application but in addition would initiate work in a fourth new area. The first Specific Aim is to demonstrate the role of intramolecular electron transfer in the production of double lesions. The investigators hypothesize that pairs of lesions separated by one, two, or possibly even more bases can result from intramolecular electron transfer processes. The second Specific Aim seeks to demonstrate conclusively that the novel lesions first identified in damaged oligomers are also produced in DNA exposed to ROS. This aim would be investigated by use of newly acquired LC-MS equipment. Crucial to this project is the applicants' archive of purified DNA oligomers containing identified ROS-induced lesions. The cell's capabilities for repairing non-adjacent double lesions are not known, nor are the mutational consequences of such lesions known. One of the lesions observed is invariably 8-oxoguanine. The third Specific Aim would be to determine which, if any, base excision repair enzymes are able to repair ROS-induced lesions. This aim has been modified in the revised application. Rather than monitor the repair in oligonucleotides containing a specific lesion, the investigators propose to monitor repair directly in ROS-exposed DNA. This work is made feasible by the use of ion-trap mass spectrometry. Finally, the investigators have added a new fourth specific aim to this revised application. It would attempt to evaluate the capacity of cells to repair base damage using cell extract and DNA substrate, again relying on ion trap technology.